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Abstract:

A methods are provided for the flow cytometry profiling of microvesicles,
including exosomes.

Claims:

1. A methods are provided for the flow cytometry profiling of
microvesicles, including exosomes.

2. The method of claim 1, wherein the profiling is performed with
nanoFACs.

3. The method of claim 2, wherein the nanoFACs utilizes a flow cytometer
tuned for maximal resolution of small particles by adding both a filter
and a small particle detector, as well as tuning the nozzle height to
eliminate drop drive noise.

4. The method of claim 1, comprising analysis of the quantity and/or
quality of microvesicles for monitoring of tumor responses to cytotoxic
therapies.

5. The method of claim 1, comprising analysis of the quantity and/or
quality of microvesicles for monitoring immune responses to tumor
vaccines.

6. The method of claim 1, comprising analysis of the quantity and/or
quality of microvesicles for monitoring immune cells following
transplantation.

7. The method of claim 1, comprising analysis of the quantity and/or
quality of microvesicles for biodosimetry to assess the level of
radiation exposure.

8. The method of claim 1, comprising utilizing a point of care device for
purposes of identifying individuals with radiation exposure or specific
infections.

9. The method of claim 1, comprising staining a population of
microvesicles with a detectably labeled affinity reagent specific for a
marker of interest, prior to analysis.

10. The method of claim 1, wherein the method further comprises selecting
a therapeutic regimen based on the analysis.

11. A kit for use in the methods of claim 1.

Description:

BACKGROUND

[0001] Exosomes are 40-150 nm vesicles secreted by a wide range of
mammalian cell types. Exosomes are one of many different sub-populations
of microvesicles that can be isolated from biofluids such as blood, urine
and cerebrospinal fluid (CSF) and from which high quality RNA and DNA can
be extracted and purified for analysis. Exosomes are shed by cells under
both normal and pathological conditions. Most exosomes studied to date
have an evolutionary-conserved set of protein molecules and a set of
tissue/cell type-specific proteins that distinguishes exosomes secreted
by different cell types. The RNA molecules in exosomes include mRNA and
miRNA, which can be shuttled from one cell to another, affecting the
recipient cell's protein production.

[0002] Exosomes are characterized in their biogenesis by formation of
intraluminal vesicles (ILVs) through the inward budding of endosomes to
form multivesicular bodies (MVBs). These MVBs then fuse with the outer
cell membrane to release their cargo of ILVs (now exosomes) to the
extracellular environment. The endosome is first formed by inward budding
of the cell membrane by endocytosis and leads to inversion of the lipid
membrane, trapping some of the extracellular environment on the
intraluminal side. Similarly, the second inward budding of the endosome
membrane traps a volume of the cell's cytoplasm and results in a positive
orientation of the ILVs lipid membrane. When the ILVs (now exosomes) are
released to the extracellular environment, they have the same orientation
as the cell membrane and have been shown to display many of the surface
markers from their cell of origin. However, the sorting process of
membrane proteins during ILV formation is an active process and thus,
exosomal surface proteins are not a simple one-to-one representation of
the surface markers from the cell of origin.

[0003] Tumors are characterized by secretion of various forms of membrane
vesicles constitutively. These comprise exosomes, MVs and apoptotic
bodies. Released membrane vesicles contain tumor-specific antigens on
their surface, e.g., Her2/Neu mesothelin, MelanA/Mart-1, CEA, HER-2, and
EGFRvIII. Furthermore, membrane vesicles from cancer cells contain RNA.
Several reports indicate that miRNA-based identification of cancer leads
to a reliable characterization of the origin and development of tumors.
As certain miRNAs are characteristic for tumors, their presence within
tumor-derived exosomes and MVs may also serve as novel biomarkers of
cancer.

[0004] Examples for key functions of exosomes include antigen presentation
and immunostimulatory and inhibitory activities. Current methods of
isolation and analytical methods include differential centrifugation and
subsequent sucrose gradient ultracentrifugation, transmission electron
microscopy (TEM), western blot and mass spectroscopy. One protocol for
exosome isolation includes ultracentrifugation and a subsequent sucrose
density gradient ultracentrifugation or, alternatively, sucrose cushion
centrifugation. However, during differential centrifugation prior to
pelleting of a given membrane vesicle population, some of the respective
vesicles may be selectively depleted. A problem of alternative protocols
is that forced filtration of membrane vesicles holds the risk of
fragmentation into smaller vesicles.

[0005] Conventionally, flow cytometry detects vesicles above approximately
˜200 nm, and therefore exosomes and smaller MVs cannot be analyzed
directly by this method. Vesicles smaller than the detection limit of the
used flow cytometer cannot be discriminated from the instrument noise,
leading to an inadequate numbering of MVs. Flow cytometry efforts for
detection of small vesicles are described by Robert et al. (2009) J
Thromb Haemost. 7(1):190-7; and Lacroix et al. (2010) J Thromb Haemost.

SUMMARY

[0006] Methods are provided for the flow cytometry profiling of
microvesicles, including exosomes; and the use of the profiling in a
variety of clinical and research applications. Antigen presenting cells
and tumor cells, among others, produce large quantities of submicron
particles, i.e. exosomes and microparticles, which modulate tumor immune
responses and the tumor microenvironment. Submicron biological particles
have been difficult to study and sort for functional studies. The present
invention provides nanoFACS, methods that allow one to analyze, sort, and
study submicron particles in functional form, without using electron
microscopy or aggregation to beads, which change the biological
properties of the particles. A cytometer was configured for maximal
resolution of small particles. Non-specific background noise was reduced
by adding both a filter and a small particle detector, as well as tuning
the nozzle height to eliminate drop drive noise.

[0007] The microvesicles are obtained from any convenient biological
sample. Serum samples from an individual are a preferred sample, which
may be treated in various ways, including binding to affinity reagents
for identification and sorting. For example, samples may be stained with
antibodies that selectively bind to markers of immune cells, tumor
markers, markers of radiation exposure, and the like. The microvesicles
may also be sorted and analyzed for the presence of nucleic acids of
interest, such as RNA, including microRNA/

[0008] Aspects of the invention include analysis of the quantity and/or
quality (for example the presence of protein or nucleic acid markers of
interest) of microvesicles for monitoring of tumor responses to cytotoxic
therapies (e.g. chemotherapy and radiation therapy). Aspects of the
invention include analysis of the quantity and/or quality (for example
the presence of protein or nucleic acid markers of interest) of
microvesicles for monitoring immune responses to tumor vaccines. Aspects
of the invention include analysis of the quantity and/or quality (for
example the presence of protein or nucleic acid markers of interest) of
microvesicles for monitoring immune cells following transplantation,
including the development of graft v host disease. Aspects of the
invention include analysis of the quantity and/or quality (for example
the presence of protein or nucleic acid markers of interest) of
microvesicles for biodosimetry, for assessing the level of radiation
exposure (e.g. from a nuclear accident, dirty bomb, etc). Such analysis
may include detecting the number of microvesicles relative to total serum
protein levels, and may include determining the presence of annexin V on
the microvesicles. Aspects of the invention include analysis of the
quantity and/or quality (for example the presence of protein or nucleic
acid markers of interest) of microvesicles which is incorporated into a
point of care device for purposes of identifying individuals with
radiation exposure or specific infections. In an embodiment, the method
further comprises assessing a clinical factor in the mammalian subject;
which may be a human subject, and combining the assessment with the
analysis of microvesicles.

[0009] In some embodiments, a patient sample, e.g. a serum sample, is
analyzed for the presence of microvesicles, which may be exosomes,
comprising markers of interest. Analysis may include mass spectroscopy,
but preferably utilizes flow cytometry with the methods of the invention.
Markers of interest include radiation specific markers, tumor specific
markers, immune cell, including antigen presenting cell such as dendritic
cell markers, and the like.

[0010] Assessment in a patient allows improved care, where patients
classified according to responsiveness can be treated with an appropriate
agent. Patients can be classified upon initial presentation of symptoms,
and can be further monitored for status over the course of the disease to
maintain appropriate therapy, or can be classified at any appropriate
stage of disease progression. Treatment of particular interest includes
radiation, e.g. including therapeutic radiation to reduce tumor size.

[0011] In other embodiments of the invention a device or kit is provided
for the analysis of patient samples. Alternatively the reagents can be
provided as a kit comprising reagents in a suspension or suspendable
form, e.g. reagents bound to beads suitable for flow cytometry, and the
like. The instructions may comprise instructions for conducting an
antibody-based flow cytometry assay.

[0012] In an embodiment, the method further comprises selecting a
therapeutic regimen based on the analysis. In an embodiment, the method
further comprises determining a treatment course for the subject based on
the analysis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office upon
request and payment of the necessary fee.

[0017]FIG. 4. Optimal FSC resolution in the nanoFACS range depends on
ability to filter the sheath fluid, addition of the optical configuration
in the BD Influx small particle option, tuning the nozzle height to
eliminate noise in the system from the drop drive, and alignment
optimization with nanoscale particles. Shown below are plots with SSC
triggering at the levels of each systems' optical noise to a SSC
background rate of <40 events/second. Conventional FACS configurations
(as with the Aria) are able to resolve nonfluorescent 400 nm beads in the
SSC dimension, but not the FSC dimension, where the nanoFACS
configuration on the Influx is able to resolve unlabeled 400 nm and 100
nm beads, as well as 40-150 nm dendritic cell exosomes above the system
SSC noise level (shown as a dashed line through the lower plots).

[0018]FIG. 5. Antibody aggregates and labeled exosomes are also able to
be resolved with nanoFACS, and this may be useful for many sorting and
fractionation needs.

DETAILED DESCRIPTION

[0019] These and other features of the present teachings will become more
apparent from the description herein. While the present teachings are
described in conjunction with various embodiments, it is not intended
that the present teachings be limited to such embodiments. On the
contrary, the present teachings encompass various alternatives,
modifications, and equivalents, as will be appreciated by those of skill
in the art.

[0020] Most of the words used in this specification have the meaning that
would be attributed to those words by one skilled in the art. Words
specifically defined in the specification have the meaning provided in
the context of the present teachings as a whole, and as are typically
understood by those skilled in the art. In the event that a conflict
arises between an art-understood definition of a word or phrase and a
definition of the word or phrase as specifically taught in this
specification, the specification shall control.

[0021] It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an," and "the" include plural
referents unless the context clearly dictates otherwise.

[0022] Compositions and methods are provided for classification and
analysis of patients having an inflammatory diseases; exposed to
radiation; cancer patients, etc. Marker signature pattern as used herein
refers to the spectrum of biomarker on microvesicles. Once the marker
levels and pattern for a particular sample are identified, the data can
be used in selecting the most appropriate therapy for an individual. By
analysis of marker levels on an individual basis, the specific subclass
of disease is determined, and the patient can be classified based on the
likelihood to respond to treatments of interest. Thus, the marker
signature can provide prognostic information to guide clinical decision
making, both in terms of institution of and escalation of therapy as well
as in the selection of the therapeutic agent to which the patient is most
likely to exhibit a robust response.

[0023] The information obtained from the marker profile is used to (a)
determine type and level of therapeutic intervention warranted (i.e. more
versus less aggressive therapy, monotherapy versus combination therapy,
type of combination therapy)), and (b) to optimize the selection of
therapeutic agents. With this approach, therapeutic regimens can be
individualized and tailored according to the specificity data obtained at
different times over the course of treatment, thereby providing a regimen
that is individually appropriate. In addition, patient samples can be
obtained at any point during the treatment process for analysis.

[0024] Mammalian species that provide samples for analysis include
canines; felines; equines; bovines; ovines; etc. and primates,
particularly humans. Animal models, particularly small mammals, e.g.
murine, lagomorpha, etc. can be used for experimental investigations.
Animal models of interest include those for models of autoimmunity, graft
rejection, and the like.

[0025] Inflammatory Disease. Inflammation is a process whereby the immune
system responds to infection or tissue damage. Inflammatory disease
results from an activation of the immune system that causes illness, in
the absence of infection or tissue damage, or at a response level that
causes illness. Inflammatory disease includes autoimmune disease, which
are any disease caused by immunity that becomes misdirected at healthy
cells and/or tissues of the body. Autoimmune diseases are characterized
by T and B lymphocytes that aberrantly target self-proteins,
-polypeptides, -peptides, and/or other self-molecules causing injury and
or malfunction of an organ, tissue, or cell-type within the body (for
example, pancreas, brain, thyroid or gastrointestinal tract) to cause the
clinical manifestations of the disease. Autoimmune diseases include
diseases that affect specific tissues as well as diseases that can affect
multiple tissues, which can depend, in part on whether the responses are
directed to an antigen confined to a particular tissue or to an antigen
that is widely distributed in the body.

[0026] The immune system employs a highly complex mechanism designed to
generate responses to protect mammals against a variety of foreign
pathogens while at the same time preventing responses against
self-antigens. In addition to deciding whether to respond (antigen
specificity), the immune system must also choose appropriate effector
functions to deal with each pathogen (effector specificity). A cell
critical in mediating and regulating these effector functions are
CD4.sup.+ T cells, which can be subtyped as TH1, TH2, TH17, etc.

[0028] The terms "subject," "individual," and "patient" are used
interchangeably herein to refer to a mammal being assessed for treatment
and/or being treated. In an embodiment, the mammal is a human. The terms
"subject," "individual," and "patient" encompass, without limitation,
individuals having cancer. Subjects may be human, but also include other
mammals, particularly those mammals useful as laboratory models for human
disease, e.g. mouse, rat, etc.

[0029] The terms "cancer," "neoplasm," and "tumor" are used
interchangeably herein to refer to cells which exhibit autonomous,
unregulated growth, such that they exhibit an aberrant growth phenotype
characterized by a significant loss of control over cell proliferation.
Cells of interest for detection, analysis, or treatment in the present
application include precancerous (e.g., benign), malignant,
pre-metastatic, metastatic, and non-metastatic cells. Cancers of
virtually every tissue are known. The phrase "cancer burden" refers to
the quantum of cancer cells or cancer volume in a subject. Reducing
cancer burden accordingly refers to reducing the number of cancer cells
or the cancer volume in a subject. The term "cancer cell" as used herein
refers to any cell that is a cancer cell or is derived from a cancer cell
e.g. clone of a cancer cell. Many types of cancers are known to those of
skill in the art, including solid tumors such as carcinomas, sarcomas,
glioblastomas, melanomas, lymphomas, myelomas, etc., and circulating
cancers such as leukemias. Examples of cancer include but are not limited
to, ovarian cancer, breast cancer, colon cancer, lung cancer, prostate
cancer, hepatocellular cancer, gastric cancer, pancreatic cancer,
cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of
the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma,
head and neck cancer, and brain cancer.

[0030] The "pathology" of cancer includes all phenomena that compromise
the well-being of the patient. This includes, without limitation,
abnormal or uncontrollable cell growth, metastasis, interference with the
normal functioning of neighboring cells, release of cytokines or other
secretory products at abnormal levels, suppression or aggravation of
inflammatory or immunological response, neoplasia, premalignancy,
malignancy, invasion of surrounding or distant tissues or organs, such as
lymph nodes, etc.

[0031] As used herein, the terms "cancer recurrence" and "tumor
recurrence," and grammatical variants thereof, refer to further growth of
neoplastic or cancerous cells after diagnosis of cancer. Particularly,
recurrence may occur when further cancerous cell growth occurs in the
cancerous tissue. "Tumor spread," similarly, occurs when the cells of a
tumor disseminate into local or distant tissues and organs; therefore
tumor spread encompasses tumor metastasis. "Tumor invasion" occurs when
the tumor growth spread out locally to compromise the function of
involved tissues by compression, destruction, or prevention of normal
organ function.

[0032] As used herein, the term "metastasis" refers to the growth of a
cancerous tumor in an organ or body part, which is not directly connected
to the organ of the original cancerous tumor. Metastasis will be
understood to include micrometastasis, which is the presence of an
undetectable amount of cancerous cells in an organ or body part which is
not directly connected to the organ of the original cancerous tumor.
Metastasis can also be defined as several steps of a process, such as the
departure of cancer cells from an original tumor site, and migration
and/or invasion of cancer cells to other parts of the body.

[0033] The term "sample" with respect to a patient encompasses blood and
other liquid samples of biological origin, solid tissue samples such as a
biopsy specimen or tissue cultures or cells derived therefrom and the
progeny thereof. The definition also includes samples that have been
manipulated in any way after their procurement, such as by treatment with
reagents; washed; or enrichment for certain cell populations, such as
cancer cells. The definition also includes sample that have been enriched
for particular types of molecules, e.g., nucleic acids, polypeptides,
etc. The term "biological sample" encompasses a clinical sample, and also
includes tissue obtained by surgical resection, tissue obtained by
biopsy, cells in culture, cell supernatants, cell lysates, tissue
samples, organs, bone marrow, blood, plasma, serum, and the like. A
"biological sample" includes a sample obtained from a patient's cancer
cell, e.g., a sample comprising polynucleotides and/or polypeptides that
is obtained from a patient's cancer cell (e.g., a cell lysate or other
cell extract comprising polynucleotides and/or polypeptides); and a
sample comprising cancer cells from a patient. A biological sample
comprising a cancer cell from a patient can also include non-cancerous
cells.

[0034] The term "diagnosis" is used herein to refer to the identification
of a molecular or pathological state, disease or condition, such as the
identification of a molecular subtype of breast cancer, prostate cancer,
or other type of cancer.

[0035] The term "prognosis" is used herein to refer to the prediction of
the likelihood of cancer-attributable death or progression, including
recurrence, metastatic spread, and drug resistance, of a neoplastic
disease, such as ovarian cancer. The term "prediction" is used herein to
refer to the act of foretelling or estimating, based on observation,
experience, or scientific reasoning. In one example, a physician may
predict the likelihood that a patient will survive, following surgical
removal of a primary tumor and/or chemotherapy for a certain period of
time without cancer recurrence.

[0036] As used herein, the terms "treatment," "treating," and the like,
refer to administering an agent, or carrying out a procedure, for the
purposes of obtaining an effect. The effect may be prophylactic in terms
of completely or partially preventing a disease or symptom thereof and/or
may be therapeutic in terms of effecting a partial or complete cure for a
disease and/or symptoms of the disease. "Treatment," as used herein, may
include treatment of a tumor in a mammal, particularly in a human, and
includes: (a) preventing the disease or a symptom of a disease from
occurring in a subject which may be predisposed to the disease but has
not yet been diagnosed as having it (e.g., including diseases that may be
associated with or caused by a primary disease; (b) inhibiting the
disease, i.e., arresting its development; and (c) relieving the disease,
i.e., causing regression of the disease.

[0037] Treating may refer to any indicia of success in the treatment or
amelioration or prevention of an cancer, including any objective or
subjective parameter such as abatement; remission; diminishing of
symptoms or making the disease condition more tolerable to the patient;
slowing in the rate of degeneration or decline; or making the final point
of degeneration less debilitating. The treatment or amelioration of
symptoms can be based on objective or subjective parameters; including
the results of an examination by a physician. Accordingly, the term
"treating" includes the administration of the compounds or agents of the
present invention to prevent or delay, to alleviate, or to arrest or
inhibit development of the symptoms or conditions associated with ocular
disease. The term "therapeutic effect" refers to the reduction,
elimination, or prevention of the disease, symptoms of the disease, or
side effects of the disease in the subject.

[0038] "In combination with", "combination therapy" and "combination
products" refer, in certain embodiments, to the concurrent administration
to a patient of a first therapeutic and the compounds as used herein.
When administered in combination, each component can be administered at
the same time or sequentially in any order at different points in time.
Thus, each component can be administered separately but sufficiently
closely in time so as to provide the desired therapeutic effect.

[0039] As used herein, the term "correlates," or "correlates with," and
like terms, refers to a statistical association between instances of two
events, where events include numbers, data sets, and the like. For
example, when the events involve numbers, a positive correlation (also
referred to herein as a "direct correlation") means that as one
increases, the other increases as well. A negative correlation (also
referred to herein as an "inverse correlation") means that as one
increases, the other decreases.

[0040] "Dosage unit" refers to physically discrete units suited as unitary
dosages for the particular individual to be treated. Each unit can
contain a predetermined quantity of active compound(s) calculated to
produce the desired therapeutic effect(s) in association with the
required pharmaceutical carrier. The specification for the dosage unit
forms can be dictated by (a) the unique characteristics of the active
compound(s) and the particular therapeutic effect(s) to be achieved, and
(b) the limitations inherent in the art of compounding such active
compound(s).

[0041] "Pharmaceutically acceptable excipient" means an excipient that is
useful in preparing a pharmaceutical composition that is generally safe,
non-toxic, and desirable, and includes excipients that are acceptable for
veterinary use as well as for human pharmaceutical use. Such excipients
can be solid, liquid, semisolid, or, in the case of an aerosol
composition, gaseous.

[0042] "Pharmaceutically acceptable salts and esters" means salts and
esters that are pharmaceutically acceptable and have the desired
pharmacological properties. Such salts include salts that can be formed
where acidic protons present in the compounds are capable of reacting
with inorganic or organic bases. Suitable inorganic salts include those
formed with the alkali metals, e.g. sodium and potassium, magnesium,
calcium, and aluminum. Suitable organic salts include those formed with
organic bases such as the amine bases, e.g., ethanolamine,
diethanolamine, triethanolamine, tromethamine, N methylglucamine, and the
like. Such salts also include acid addition salts formed with inorganic
acids (e.g., hydrochloric and hydrobromic acids) and organic acids (e.g.,
acetic acid, citric acid, maleic acid, and the alkane- and arene-sulfonic
acids such as methanesulfonic acid and benzenesulfonic acid).
Pharmaceutically acceptable esters include esters formed from carboxy,
sulfonyloxy, and phosphonoxy groups present in the compounds, e.g.,
C1-6 alkyl esters. When there are two acidic groups present, a
pharmaceutically acceptable salt or ester can be a mono-acid-mono-salt or
ester or a di-salt or ester; and similarly where there are more than two
acidic groups present, some or all of such groups can be salified or
esterified. Compounds named in this invention can be present in
unsalified or unesterified form, or in salified and/or esterified form,
and the naming of such compounds is intended to include both the original
(unsalified and unesterified) compound and its pharmaceutically
acceptable salts and esters. Also, certain compounds named in this
invention may be present in more than one stereoisomeric form, and the
naming of such compounds is intended to include all single stereoisomers
and all mixtures (whether racemic or otherwise) of such stereoisomers.

[0043] The terms "pharmaceutically acceptable", "physiologically
tolerable" and grammatical variations thereof, as they refer to
compositions, carriers, diluents and reagents, are used interchangeably
and represent that the materials are capable of administration to or upon
a human without the production of undesirable physiological effects to a
degree that would prohibit administration of the composition.

[0044] A "therapeutically effective amount" means the amount that, when
administered to a subject for treating a disease, is sufficient to effect
treatment for that disease.

[0045] "Suitable conditions" shall have a meaning dependent on the context
in which this term is used. That is, when used in connection with an
antibody, the term shall mean conditions that permit an antibody to bind
to its corresponding antigen. When used in connection with contacting an
agent to a cell, this term shall mean conditions that permit an agent
capable of doing so to enter a cell and perform its intended function. In
one embodiment, the term "suitable conditions" as used herein means
physiological conditions.

[0046] The term "inflammatory" response is the development of a humoral
(antibody mediated) and/or a cellular (mediated by antigen-specific T
cells or their secretion products) response. An "immunogen" is capable of
inducing an immunological response against itself on administration to a
mammal or due to autoimmune disease.

[0047] The terms "biomarker," "biomarkers," "marker" or "markers" refer
to, without limitation, cytokines, chemokines, growth factors, proteins,
peptides, nucleic acids, oligonucleotides, and metabolites, together with
their related metabolites, mutations, variants, polymorphisms,
modifications, fragments, subunits, degradation products, elements, and
other analytes or sample-derived measures. Markers can also include
mutated proteins, mutated nucleic acids, variations in copy numbers
and/or transcript variants. Markers also encompass non-blood borne
factors and non-analyte physiological markers of health status, and/or
other factors or markers not measured from samples (e.g., biological
samples such as bodily fluids), such as clinical parameters and
traditional factors for clinical assessments. Markers can also include
any indices that are calculated and/or created mathematically. Markers
can also include combinations of any one or more of the foregoing
measurements, including temporal trends and differences.

[0048] To "analyze" includes determining a set of values associated with a
sample by measurement of a marker (such as, e.g., presence or absence of
a marker or constituent expression levels) in the sample and comparing
the measurement against measurement in a sample or set of samples from
the same subject or other control subject(s). The markers of the present
teachings can be analyzed by any of various conventional methods known in
the art. To "analyze" can include performing a statistical analysis to,
e.g., determine whether a subject is a responder or a non-responder to a
therapy.

[0049] A "sample" in the context of the present teachings refers to any
biological sample that is isolated from a subject. A sample can include,
without limitation an aliquot of body fluid, whole blood, serum, plasma,
tissue biopsies, synovial fluid, lymphatic fluid, ascites fluid, and
interstitial or extracellular fluid. The term "sample" also encompasses
the fluid in spaces between cells, including gingival crevicular fluid,
bone marrow, cerebrospinal fluid (CSF), saliva, mucous, sputum, semen,
sweat, urine, or any other bodily fluids. "Blood sample" can refer to
whole blood or any fraction thereof, including serum and plasma. Samples
can be obtained from a subject by means including but not limited to
venipuncture, excretion, ejaculation, massage, biopsy, needle aspirate,
lavage, scraping, surgical incision, or intervention or other means known
in the art.

[0050] A "dataset" is a set of numerical values resulting from evaluation
of a sample (or population of samples) under a desired condition. The
values of the dataset can be obtained, for example, by experimentally
obtaining measures from a sample and constructing a dataset from these
measurements; or alternatively, by obtaining a dataset from a service
provider such as a laboratory, or from a database or a server on which
the dataset has been stored. Similarly, the term "obtaining a dataset
associated with a sample" encompasses obtaining a set of data determined
from at least one sample. Obtaining a dataset encompasses obtaining a
sample, and processing the sample to experimentally determine the data,
e.g., via measuring, PCR, microarray, one or more primers, one or more
probes, antibody binding, or ELISA. The phrase also encompasses receiving
a set of data, e.g., from a third party that has processed the sample to
experimentally determine the dataset. Additionally, the phrase
encompasses mining data from at least one database or at least one
publication or a combination of databases and publications.

[0051] "Measuring" or "measurement" in the context of the present
teachings refers to determining the presence, absence, quantity, amount,
or effective amount of a substance in a clinical or subject-derived
sample, including the presence, absence, or concentration levels of such
substances, and/or evaluating the values or categorization of a subject's
clinical parameters based on a control.

[0052] Classification can be made according to predictive modeling methods
that set a threshold for determining the probability that a sample
belongs to a given class. The probability preferably is at least 50%, or
at least 60% or at least 70% or at least 80% or higher. Classifications
also can be made by determining whether a comparison between an obtained
dataset and a reference dataset yields a statistically significant
difference. If so, then the sample from which the dataset was obtained is
classified as not belonging to the reference dataset class. Conversely,
if such a comparison is not statistically significantly different from
the reference dataset, then the sample from which the dataset was
obtained is classified as belonging to the reference dataset class.

[0053] The predictive ability of a model can be evaluated according to its
ability to provide a quality metric, e.g. AUC or accuracy, of a
particular value, or range of values. In some embodiments, a desired
quality threshold is a predictive model that will classify a sample with
an accuracy of at least about 0.7, at least about 0.75, at least about
0.8, at least about 0.85, at least about 0.9, at least about 0.95, or
higher. As an alternative measure, a desired quality threshold can refer
to a predictive model that will classify a sample with an AUC (area under
the curve) of at least about 0.7, at least about 0.75, at least about
0.8, at least about 0.85, at least about 0.9, or higher.

[0054] As is known in the art, the relative sensitivity and specificity of
a predictive model can be "tuned" to favor either the selectivity metric
or the sensitivity metric, where the two metrics have an inverse
relationship. The limits in a model as described above can be adjusted to
provide a selected sensitivity or specificity level, depending on the
particular requirements of the test being performed. One or both of
sensitivity and specificity can be at least about at least about 0.7, at
least about 0.75, at least about 0.8, at least about 0.85, at least about
0.9, or higher.

[0055] Unless otherwise apparent from the context, all elements, steps or
features of the invention can be used in any combination with other
elements, steps or features.

[0057] The invention has been described in terms of particular embodiments
found or proposed by the present inventor to comprise preferred modes for
the practice of the invention. It will be appreciated by those of skill
in the art that, in light of the present disclosure, numerous
modifications and changes can be made in the particular embodiments
exemplified without departing from the intended scope of the invention.
Due to biological functional equivalency considerations, changes can be
made in protein structure without affecting the biological action in kind
or amount. All such modifications are intended to be included within the
scope of the appended claims.

[0058] The subject methods are used for prophylactic or therapeutic
purposes. As used herein, the term "treating" is used to refer to both
prevention of relapses, and treatment of pre-existing conditions. For
example, the prevention of inflammatory disease can be accomplished by
administration of the agent prior to development of a relapse. The
treatment of ongoing disease, where the treatment stabilizes or improves
the clinical symptoms of the patient, is of particular interest.

Methods

[0059] A sample from an individual is analyzed for the presence of
microvesicles, which are optionally detectable labeled for one or more
markers of interest. Parameters of interest include microvesicle size,
quantity, presence of RNA of interest, presence of proteins of interest,
presence of lipids of interest.

[0060] Flow cytometry may be used in the analysis and sorting of the
vesicles. FACS fluidics configurations include, in addition to routine
0.22 μm prefiltering for sheath fluid, inline filters of from about
0.02 to about 0.1 μm to minimize particulate noise. Filters of
interest may be from about 0.05 to about 0.1 μm, although sheath
pressure may be increased to deliver stable pressure. The flow cytometry
optical configurations are typically also adjusted for the small
particles, using a high magnification lens, which images the scattered
stream on a pinhole for detection of low noise detection of small signals
while preserving linearity for detecting large signals.

[0061] The signature pattern can be generated from a biological sample
using any convenient protocol. The readout can be a mean, average, median
or the variance or other statistically or mathematically-derived value
associated with the measurement. The marker readout information can be
further refined by direct comparison with the corresponding reference or
control pattern. A binding pattern can be evaluated on a number of
points: to determine if there is a statistically significant change at
any point in the data matrix; whether the change is an increase or
decrease in the binding; whether the change is specific for one or more
physiological states, and the like. The absolute values obtained for each
marker under identical conditions will display a variability that is
inherent in live biological systems and also reflects the variability
inherent between individuals.

[0062] Following obtainment of the signature pattern from the sample being
assayed, the signature pattern is compared with a reference or control
profile to make a prognosis regarding the phenotype of the patient from
which the sample was obtained/derived. Typically a comparison is made
with a sample or set of samples from an unaffected, normal source.
Additionally, a reference or control signature pattern can be a signature
pattern that is obtained from a sample of a patient known to be
responsive or non-responsive to the therapy of interest, and therefore
can be a positive reference or control profile.

[0063] In certain embodiments, the obtained signature pattern is compared
to a single reference/control profile to obtain information regarding the
phenotype of the patient being assayed. In yet other embodiments, the
obtained signature pattern is compared to two or more different
reference/control profiles to obtain more in depth information regarding
the phenotype of the patient. For example, the obtained signature pattern
can be compared to a positive and negative reference profile to obtain
confirmed information regarding whether the patient has the phenotype of
interest.

[0064] The detection reagents can be provided as part of a kit. Thus, the
invention further provides kits for detecting the presence of a panel of
specific markers of interest in a biological sample. Procedures using
these kits can be performed by clinical laboratories, experimental
laboratories, medical practitioners, or private individuals. The kits of
the invention for detecting markers comprise affinity reagents useful for
generating a prognostic signature pattern, which can be provided in
solution or bound to a substrate. The kit can optionally provide
additional components that are useful in the procedure, including, but
not limited to, buffers, developing reagents, labels, reacting surfaces,
means for detection, control samples, standards, instructions, and
interpretive information.

[0065] In addition to the above components, the subject kits will further
include instructions for practicing the subject methods. These
instructions can be present in the subject kits in a variety of forms,
one or more of which can be present in the kit. One form in which these
instructions can be present is as printed information on a suitable
medium or substrate, e.g., a piece or pieces of paper on which the
information is printed, in the packaging of the kit, in a package insert,
etc. Yet another means would be a computer readable medium, e.g.,
diskette, CD, hard-drive, network data storage, etc., on which the
information has been recorded. Yet another means that can be present is a
website address which can be used via the internet to access the
information at a removed site. Any convenient means can be present in the
kits.

Assessment of Patient Outcomes

[0066] Patient outcomes and status can be assessed using imaging-based
criteria such as radiographic scores, clinical and laboratory criteria.
Multiple different imaging, clinical and laboratory criteria and scoring
systems have been and are being developed to assess disease activity and
response to therapy in cancer, radiation exposure, and inflammatory
diseases, etc.

[0067] A pattern can be obtained as a dataset for an indication of
interest. The dataset comprises quantitative data for the presence in
serum of at least 1 microvesicle marker, etc. The dataset optionally
quantitative data for the presence in a clinical sample of other markers,
including immune cell presence or specificity, clinical indices, and the
like. A statistical test will provide a confidence level for a change in
the expression, titers or concentration of markers between the test and
control profiles to be considered significant, where the control profile
can be for selected as appropriate. The raw data can be initially
analyzed by measuring the values for each marker, usually in duplicate,
triplicate, quadruplicate or in 5-10 replicate features per marker.

[0068] A test dataset is considered to be different than a control dataset
if one or more of the parameter values of the profile exceeds the limits
that correspond to a predefined level of significance.

[0069] To provide significance ordering, the false discovery rate (FDR)
can be determined. First, a set of null distributions of dissimilarity
values is generated. In one embodiment, the values of observed profiles
are permuted to create a sequence of distributions of correlation
coefficients obtained out of chance, thereby creating an appropriate set
of null distributions of correlation coefficients (see Tusher et al.
(2001) PNAS 98, 5116-21, herein incorporated by reference). This analysis
algorithm is currently available as a software "plug-in" for Microsoft
Excel know as Significance Analysis of Microarrays (SAM). The set of null
distribution is obtained by: permuting the values of each profile for all
available profiles; calculating the pair-wise correlation coefficients
for all profile; calculating the probability density function of the
correlation coefficients for this permutation; and repeating the
procedure for N times, where N is a large number, usually 300. Using the
N distributions, one calculates an appropriate measure (mean, median,
etc.) of the count of correlation coefficient values that their values
exceed the value (of similarity) that is obtained from the distribution
of experimentally observed similarity values at given significance level.

[0070] The FDR is the ratio of the number of the expected falsely
significant correlations (estimated from the correlations greater than
this selected Pearson correlation in the set of randomized data) to the
number of correlations greater than this selected Pearson correlation in
the empirical data (significant correlations). This cut-off correlation
value can be applied to the correlations between experimental profiles.

[0071] For SAM, Z-scores represent another measure of variance in a
dataset, and are equal to a value of X minus the mean of X, divided by
the standard deviation. A Z-Score tells how a single data point compares
to the normal data distribution. A Z-score demonstrates not only whether
a datapoint lies above or below average, but how unusual the measurement
is. The standard deviation is the average distance between each value in
the dataset and the mean of the values in the dataset.

[0072] Using the aforementioned distribution, a level of confidence is
chosen for significance. This is used to determine the lowest value of
the correlation coefficient that exceeds the result that would have
obtained by chance. Using this method, one obtains thresholds for
positive correlation, negative correlation or both. Using this
threshold(s), the user can filter the observed values of the pairwise
correlation coefficients and eliminate those that do not exceed the
threshold(s). Furthermore, an estimate of the false positive rate can be
obtained for a given threshold. For each of the individual "random
correlation" distributions, one can find how many observations fall
outside the threshold range. This procedure provides a sequence of
counts. The mean and the standard deviation of the sequence provide the
average number of potential false positives and its standard deviation.

[0073] The data can be subjected to non-supervised hierarchical clustering
to reveal relationships among profiles. For example, hierarchical
clustering can be performed, where the Pearson correlation is employed as
the clustering metric. One approach is to consider a patient disease
dataset as a "learning sample" in a problem of "supervised learning".
CART is a standard in applications to medicine (Singer (1999) Recursive
Partitioning in the Health Sciences, Springer), which can be modified by
transforming any qualitative features to quantitative features; sorting
them by attained significance levels, evaluated by sample reuse methods
for Hotelling's T2 statistic; and suitable application of the lasso
method. Problems in prediction are turned into problems in regression
without losing sight of prediction, indeed by making suitable use of the
Gini criterion for classification in evaluating the quality of
regressions.

[0074] Other methods of analysis that can be used include logic
regression. One method of logic regression Ruczinski (2003) Journal of
Computational and Graphical Statistics 12:475-512. Logic regression
resembles CART in that its classifier can be displayed as a binary tree.
It is different in that each node has Boolean statements about features
that are more general than the simple "and" statements produced by CART.

[0075] Another approach is that of nearest shrunken centroids (Tibshirani
(2002) PNAS 99:6567-72). The technology is k-means-like, but has the
advantage that by shrinking cluster centers, one automatically selects
features (as in the lasso) so as to focus attention on small numbers of
those that are informative. The approach is available as Prediction
Analysis of Microarrays (PAM) software, a software "plug-in" for
Microsoft Excel, and is widely used. Two further sets of algorithms are
random forests (Breiman (2001) Machine Learning 45:5-32 and MART (Hastie
(2001) The Elements of Statistical Learning, Springer). These two methods
are already "committee methods." Thus, they involve predictors that
"vote" on outcome. Several of these methods are based on the "R"
software, developed at Stanford University, which provides a statistical
framework that is continuously being improved and updated in an ongoing
basis.

[0077] These tools and methods can be applied to several classification
problems. For example, methods can be developed from the following
comparisons: i) all cases versus all controls, ii) all cases versus
post-radiation exposure controls, iii) all cases versus non-exposed
controls.

[0078] These statistical tools are applicable to all manner of marker
data. A set of data that can be easily determined, and that is highly
informative regarding detection of individuals with clinically
significant responsiveness to therapy, exposure to radiation, etc. is
provided.

[0079] Also provided are databases of signature patterns for patient
status. Such databases will typically comprise signature patterns of
individuals having phenotypes such as responsive, post-radiation, etc.,
where such profiles are as described above.

[0080] The analysis and database storage can be implemented in hardware or
software, or a combination of both. In one embodiment of the invention, a
machine-readable storage medium is provided, the medium comprising a data
storage material encoded with machine readable data which, when using a
machine programmed with instructions for using said data, is capable of
displaying a any of the datasets and data comparisons of this invention.
Such data can be used for a variety of purposes, such as patient
monitoring, initial diagnosis, and the like. Preferably, the invention is
implemented in computer programs executing on programmable computers,
comprising a processor, a data storage system (including volatile and
non-volatile memory and/or storage elements), at least one input device,
and at least one output device. Program code is applied to input data to
perform the functions described above and generate output information.
The output information is applied to one or more output devices, in known
fashion. The computer can be, for example, a personal computer,
microcomputer, or workstation of conventional design.

[0081] Each program is preferably implemented in a high level procedural
or object oriented programming language to communicate with a computer
system. However, the programs can be implemented in assembly or machine
language, if desired. In any case, the language can be a compiled or
interpreted language. Each such computer program is preferably stored on
a storage media or device (e.g., ROM or magnetic diskette) readable by a
general or special purpose programmable computer, for configuring and
operating the computer when the storage media or device is read by the
computer to perform the procedures described herein. The system can also
be considered to be implemented as a computer-readable storage medium,
configured with a computer program, where the storage medium so
configured causes a computer to operate in a specific and predefined
manner to perform the functions described herein.

[0082] A variety of structural formats for the input and output means can
be used to input and output the information in the computer-based systems
of the present invention. One format for an output means test datasets
possessing varying degrees of similarity to a trusted profile. Such
presentation provides a skilled artisan with a ranking of similarities
and identifies the degree of similarity contained in the test pattern.

[0083] The signature patterns and databases thereof can be provided in a
variety of media to facilitate their use. "Media" refers to a manufacture
that contains the signature pattern information of the present invention.
The databases of the present invention can be recorded on computer
readable media, e.g. any medium that can be read and accessed directly by
a computer. Such media include, but are not limited to: magnetic storage
media, such as floppy discs, hard disc storage medium, and magnetic tape;
optical storage media such as CD-ROM; electrical storage media such as
RAM and ROM; and hybrids of these categories such as magnetic/optical
storage media. One of skill in the art can readily appreciate how any of
the presently known computer readable mediums can be used to create a
manufacture comprising a recording of the present database information.
"Recorded" refers to a process for storing information on computer
readable medium, using any such methods as known in the art. Any
convenient data storage structure can be chosen, based on the means used
to access the stored information. A variety of data processor programs
and formats can be used for storage, e.g. word processing text file,
database format, etc.

Clinical Factors

[0084] In some embodiments, one or more clinical factors in a subject can
be assessed. In some embodiments, assessment of one or more clinical
factors in a subject can be combined with a marker analysis in the
subject to identify status of the subject.

[0085] Various clinical factors are generally known one of ordinary skill
in the art to be associated with the disease in question. In some
embodiments, clinical factors known to one of ordinary skill in the art
to be associated with the disease, can include age, gender, race, family
history, and/or medications. In some embodiments, a clinical factor can
include age at onset of disease, duration of therapeutic treatment,
and/or the relapse rate of the subject.

[0086] It is to be understood that this invention is not limited to the
particular methodology, protocols, cell lines, animal species or genera,
and reagents described, as such may vary. It is also to be understood
that the terminology used herein is for the purpose of describing
particular embodiments only, and is not intended to limit the scope of
the present invention which will be limited only by the appended claims.

[0087] The following examples are put forth so as to provide those of
ordinary skill in the art with a complete disclosure and description of
how to make and use the subject invention, and are not intended to limit
the scope of what is regarded as the invention. Efforts have been made to
ensure accuracy with respect to the numbers used (e.g. amounts,
temperature, concentrations, etc.) but some experimental errors and
deviations should be allowed for. Unless otherwise indicated, parts are
parts by weight, molecular weight is average molecular weight,
temperature is in degrees centigrade; and pressure is at or near
atmospheric.

[0088] All publications and patent applications cited in this
specification are herein incorporated by reference as if each individual
publication or patent application were specifically and individually
indicated to be incorporated by reference.

EXPERIMENTAL

Example 1

[0089] NanoFACS has been developed as a method for studying submicron
particles in blood samples.

[0090] These submicron particles are associated with numerous biological
conditions, and subsets and profiles of these particles are useful as
minimally invasive biomarkers for monitoring immune responses, including
the development of tumor immunity; tumor stage and treatment responses or
progression; and radiation exposure.

[0091] Circulating submicron particles are being studied in many fields of
medicine as biomarkers and mediators of disease. However, cytometric
separation and functional studies of these particles have been limited by
the small and overlapping sizes of these cell-derived particles.
Exosomes, microvesicles, and apoptotic blebs represent morphologically
and functionally distinct populations of submicron membrane-bound
particles. Exosomes, which are formed in microvesicular bodies, secreted
in a programmed manner, and measure 40-150 nm in diameter, are
functionally and morphologically distinct from microparticles (100-1000
nm) that are shed by cells in response to stressors and stimuli, and
distinct from apoptotic blebs (>800nm) that detach from dying cells
early after induction of apoptosis.

[0092] Tumors, antigen presenting cells, and platelets produce especially
large quantities of exosomes and circulating microparticles, with
distinct surface receptor and miRNA repertoires, that have wide ranges of
cellular and physiological effects, including malignant progression,
immune modulation, and thrombosis. Although the biological effects of
submicron particles are significant, size has been a barrier to
functional sorting and analysis of subsets of these particles. In order
to be able to fractionate submicron particles in functional forms, based
on size and receptor staining, we developed a method for sorting these
particles with nano-scale Fluorescence Activated Cell Sorting (nanoFACS).
FACS has been widely used, but sorting of 20-400 nm submicron
subpopulations has not been demonstrated previously.

[0093] To detect and sort subsets of exosomes and other submicron
particles, we configured an Influx flow cytometer (BD Biosciences, San
Jose, Calif.) for maximal resolution of small particles. Non-specific
background noise was reduced by adding a 0.02 or 0.1 micron filter in the
sheath fluid line close to the nozzle. A PMT and high magnification
collection lens was added to the Forward Scatter (FSc) channel to
increase sensitivity. Although FSc is conventionally used as a trigger
parameter, we find that noise triggers in the FSc and SSc channels in our
system overlap with the scatter signals from 200 nm and 100 nm particles,
respectively. Thus, for discriminating and sorting unlabeled 100-1000 nm
particles, SSc was used as a trigger signal. Lastly, the sorting
conditions (drop drive amplitude and phase) were set so there was no
increase in the sheath trigger rate. With this configuration, 100 nm
polystyrene beads (Spherotech and Invitrogen) are detected and are
sortable at and above the level of background SSc noise. If a fluorescent
trigger is used, it is possible to measure and sort particles as small as
40 nm. 20-40 nm fluorescent particles demonstrate overlapping
distributions detectable above the fluorescent threshold.

[0094] To demonstrate the utility of nanoFACS for fractionating distinct
sub-micron sub-populations of biological interest, FSc vs SSC profiles of
supernatants from irradiated dendritic cells were gated and sorted based
on FSC gating. Counterstaining with CD9 and class I MHC APC-conjugated
antibodies suggested that the smaller particles were DC2.4-derived
exosomes. The largest particles were annexin V-FITC positive, consistent
with apoptotic blebs or microparticles. The sorted populations
demonstrated distinct morphological profiles by electron microscopy,
consistent with the staining patterns measured. Diffusion light
scattering and nanoparticle tracking analysis (DLS-NTA, Nanosight LM-10)
gave further confirmation of the size distribution of the sorted
populations.

[0095] NanoFACS extends the range of FACS-based single particle
characterization and sorting by an order of magnitude. Sorting subsets of
exosomes, microparticles, viruses, and other 40-1000 nm particles with
nanoFACS will be useful in many fields of medicine for diagnostic assays,
functional studies, and therapeutic enrichment or depletion.

[0096] Identification of unique exosome and microparticle profiles and
subsets is clinically useful in many fields, and our focus is on
developing these biomarkers for use in the fields of immunology,
oncology, and biodefense. Identification of tumor- and immune
cell-specific markers enables the use of submicron particles circulating
in the serum to monitor tumor and immune cellular status. Combining these
markers with radiation-specific markers enables monitoring the
intratumoral microenvironment noninvasively. Additional applications in
cardiology, hematology, infectious disease, and critical care also have
rapid translational potential.

[0097] Tumor Immune Response Monitoring: For identification of exosome and
microparticle profiles associated with anti-tumor immunity, there are
well established mouse models of effective vs. suppressive immune
responses. Plasma is sampled during the development of these immune
responses to define submicron particle profiles that are positively (as
with allogeneic tumor rejection) versus negatively associated (as with
the development of immunosuppressive effects in the 4T1 breast cancer
model) with anti-tumor immune responses. Treatment responses can be
associated with distinct exosome profiles.

[0099] Radiation Response/Exposure Monitoring: In mice and humans treated
with total body or localized tumor irradiation, data that shows distinct
particle profiles. For example, increased microparticles in plasma are
found from patients treated with a single large dose of localized
irradiation for locally advanced pancreatic cancer.

Example 2

[0100] To demonstrate the utility of this method for fractionating
distinct sub-popultations, we examined biological fluids with
unfractionated submicron particle populations and sorted separate exosome
and microparticle populations. In serum and in dendritic cell cultures,
exosomes predominate. DC2.4-derived exosomes were doubly positive for CD9
and class I MHC, with minimal annexin V staining. Microparticles, in
contrast, are characterized by exposed phosphatidyl serime and were
annexin V positive. We sorted these populations and demonstrated distinct
morphological profiles by electron microscopy and confirmed
nanoFACS-sorted particle sizes with diffusion light scattering
nanoparticle DLS-NTA.

[0101] Characterization and sorting at a single particle level offers
several advantages over currently available methods, which analyze of
exosomes and other microparticles in bulk (unsorted) or as bead-bound
aggregates. FACS has been a critical tool for determining cell types,
functions, and lineages in immunology, stem cell biology, and
microbiology. NanoFACS is useful for sorting subsets of 40-1000 nm
particles, including exosomes, microparticles, viruses, and microbes, for
diagnostic and functional studies that were not previously feasible.
NanoFACS will benefit a diverse group of scientists studying nano-scale
biological particles in fields as wide ranging as medicine, biodefense,
and marine biology. Particles that can be analyzed and sorted by the
methods of the invention include:

[0102] FACS fluidics configurations: In addition to the use of routine
0.22 μm prefiltering for sheath fluid, we tested inline filters 0.02
and 0.1 um to minimize particulate noise in the sheath fluid. Use of a
0.1 μm filter in the sheath line close to the nozzle eliminates
>99% of the detectable particulate debris. With the 0.1 um filter, it
was necessary to increases the sheath pressure from 20 psi to 23 psi to
deliver the same pressure to the nozzle, and consistent sheath flow rates
were more stable with 0.1 instead of 0.02 um filtering.

[0103] SSC trigger vs. FL-1 trigger. By triggering on the fluorescent
signal, we determined the proportion of particles below the SSC threshold
by determining the ratio of particles identified by fluorescent labeling
above and below the SSC SSC-488 threshold.

[0104] Flow cytometry optical configurations: A BD Influx flow cytometer
was configured with a high magnification lens (20×), with images
the scattered stream on a 0.7 mm pinhole. Light passing the pinhole is
detected by a PMT for detection of low noise detection of small signals,
while preserving linearity for detecting large signals. Variances were
compared for the FSC and SSC channels, to confirm superior small particle
size resolution with SSC. SSC and FSc gains were adjusted to place 400 nm
particles near the top of the scale, and the SSC threshold was adjusted
for a count rate on sheath fluid of 20-70 events/sec with the drop drive
off.